Cyperusrotundus is an important medicinal plant widely used in traditional medicine around the world. An in vitro tissue culture of this plant was raised for investigating its naturally occurring insecticidal compound- the rotenoids. Callus of C. rotunduswas initiated from young rhizomes on MS basal medium supplemented with different concentrations of growth hormones. Five rotenoids viz., elliptone, tephrosin, rotenone, sumatrol and deguelin were identified from plant parts (rhizome and leaves) as well as callus cultures on the basis of  thin layer-chromatography, Gas liquid-chromatography and high performance liquid -chromatography. Rotenoid content was higherin rhizome (6.7 mg/g.dw) in comparison to leaf and calli (4.6 mg/g.dw and 6.2 mg/g.dw respectively).